The NADPH-oxidase Nox1 generates reactive oxygen species (ROS) that function through various signaling pathways to activate mitogenic growth and angiogenesis. Nox1 is overexpressed in ~ 60-70% of early human colon cancers, and considerable data point to a role in tumorigenesis. Introduction of mutationally activated K-Ras results in marked induction of Nox1 mRNA, and K-Ras-induced cell growth is prevented by antioxidants and by siRNA to Nox1. We propose that overexpression of Nox1 in human colon cancers can result from activation of Nox1 transcription by RasV12. This grant proposes to investigate the correlation of mutationally activated K-Ras and Nox1 overexpression in human tumor samples, and to investigate the signaling pathways leading to transcriptional activation of Nox1 expression. To evaluate the causal relationship between K-Ras oncogenic mutation, Nox1 overexpression, and intestinal neoplasia, Nox1 overexpressing and Nox1 knockout mice have been developed. These will also be used to test the hypothesis that Nox1 plays a causal role in intestinal tumors induced in a genetic background consisting of mutationally inactivated APC and mutationally activated K-Ras. These studies are providing proof-of-concept data for Nox1 as a therapeutic target. In a newly proposed aim, we will use medicinal chemistry approaches to improve inhibitors of Nox1 that we identified during high throughput screening of a Nox1 regulatory protein. Novel strategies are presented to improve affinity as well as isoform selectivity, so as to develop inhibitors that individually target Nox1 and Nox2. These inhibitors will be used to augment cell biological approaches in the original aims, and will also be evaluated for their potential as Nox-directed drugs. These studies will provide novel information regarding the role of Nox1 in GI cancers and may provide an important new treatment modality. PUBLIC HEALTH RELEVANCE: Free radicals that are made from oxygen have recently been found to be overproduced in several types of cancers, including those of the digestive tract. The major source of these radicals has recently been discovered to be a type of enzyme called a "Nox", and one of these, Nox1, is overproduced in colon cancer. This grant studies the way in which the gene for Nox1 is turned on to overproduce oxygen radicals and the role of these radicals as signals that cause cancer cells to divide and recruit new blood vessels. A new goal of the grant is to develop inhibitor molecules that will act on Nox1 to turn off the production of these free radicals. Such inhibitors will have potential in the treatment of several types of diseases, including cancer, cardiovascular diseases, and shock lung.